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Autocamtide 2, amide

Cat No.:V77226 Purity: ≥98%
Autocamtide 2, amide is a substrate for calmodulin kinase (CaMK) family experiments (100 μM final concentration).
Autocamtide 2, amide
Autocamtide 2, amide Chemical Structure Product category: Autophagy
This product is for research use only, not for human use. We do not sell to patients.
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1mg
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Product Description
Autocamtide 2, amide is a substrate for calmodulin kinase (CaMK) family experiments (100 μM final concentration).
Autocamtide 2, amide is a synthetic peptide amide that serves as a highly selective substrate for calcium/calmodulin‑dependent protein kinase II (CaMKII). The amidated C‑terminus improves peptide stability by reducing proteolytic degradation from carboxypeptidases. Autocamtide‑2 is widely used in kinase activity assays to measure CaMKII activation in complex biological samples.
Biological Activity I Assay Protocols (From Reference)
Targets
CaMK
Autocamtide‑2 targets the substrate‑binding site of CaMKII and is phosphorylated by CaMKII on a specific serine residue. The peptide contains the CaMKII substrate consensus sequence (RXXS/T) and has high selectivity for CaMKII over other protein kinases, including PKA, PKC, CaMKIV and CaMKI. Autocamtide‑2 is a more selective substrate than syntide‑2, making it the preferred tool for measuring CaMKII activity.
ln Vitro
In cell‑free kinase assays, autocamtide‑2 amide is phosphorylated by recombinant CaMKII in a Ca2+/calmodulin‑dependent manner. The Km value for CaMKII is typically 10‑20 uM, with a kcat of 5‑10 s-¹. The peptide is not significantly phosphorylated by PKA, PKC, CaMKIV or CaMKI at concentrations up to 100 uM. The amide modification does not alter the substrate kinetics compared to the free acid form but provides enhanced stability in cell lysates.
ln Vivo
No direct in vivo activity is described, as autocamtide‑2 is a substrate rather than a therapeutic agent. However, it is used in ex vivo kinase activity assays to measure CaMKII activation in tissue lysates from animal models of neurological disorders (e.g., Alzheimer's disease, LTP induction), cardiac disease (e.g., hypertrophy, ischaemia), and cancer. The peptide helps to elucidate the role of CaMKII in synaptic plasticity, cardiac contractility and tumour progression.
Enzyme Assay
A standard CaMKII kinase assay (radioactive): Autocamtide‑2 amide (final concentration 10‑100 uM) is incubated with recombinant CaMKII (5‑20 nM) in assay buffer (50 mM HEPES pH 7.4, 10 mM MgCl2, 0.5 mM CaCl2, 1 uM calmodulin, 1 mM DTT, 0.1 mg/mL BSA) for 5‑10 min at 30degC. The reaction is initiated by adding 100 uM ATP (with 0.5 uCi/well 33P‑ATP). After 5‑20 min, the reaction is terminated by spotting onto P81 phosphocellulose paper, washed 3 times with 0.75% phosphoric acid, and counted by liquid scintillation. Controls without Ca2+/calmodulin or with EGTA (2 mM) determine Ca2+‑independent activity. IC50 values for CaMKII inhibitors can be calculated by adding inhibitors to the reaction mix.
Cell Assay
A general cellular CaMKII activity assay (ex vivo): Cells or tissue samples (e.g., primary neurons, cardiac myocytes, brain tissue lysates) are lysed in RIPA buffer containing phosphatase and protease inhibitors. Protein concentration is normalised (0.5‑1 ug/uL). Autocamtide‑2 amide (50 uM) is added to the lysate along with Ca2+/calmodulin (0.5 mM CaCl2, 1 uM calmodulin) and ATP (100 uM) in assay buffer. The reaction is incubated at 30degC for 30 min. Phosphorylation is quantified by transferring the reaction to a streptavidin‑coated plate (if biotinylated peptide is used) and detecting with an anti‑phospho‑specific antibody or by measuring incorporated 33P‑ATP. CaMKII activity is expressed as pmol phosphate incorporated per mg protein per minute.
Animal Protocol
A general animal protocol for CaMKII activity measurement: Male C57BL/6 mice or Sprague‑Dawley rats are subjected to treatments that modulate CaMKII activity (e.g., induction of long‑term potentiation (LTP) by electrical stimulation, ischaemia‑reperfusion injury, cardiac pressure overload by transverse aortic constriction). Animals are euthanised by cervical dislocation or isoflurane overdose. Brain (hippocampus, cortex) or cardiac tissue is rapidly dissected, snap‑frozen in liquid nitrogen, and stored at -80degC. For activity measurement, tissue is homogenised in lysis buffer and processed as described in the cellular assay protocol. Enzyme activity is normalised to total protein content (BCA assay) or to total CaMKII protein level (western blot).
ADME/Pharmacokinetics
As a peptide substrate, autocamtide‑2 amide has a short plasma half‑life (minutes) due to rapid proteolytic degradation and is not used as a therapeutic agent. For ex vivo assays, the peptide is added directly to tissue lysates, so pharmacokinetic properties are not applicable. The amide modification reduces cleavage by carboxypeptidases but does not affect absorption or distribution. For storage, the lyophilised powder should be kept at -20degC or -80degC, protected from moisture and light.
Toxicity/Toxicokinetics
Autocamtide‑2 amide is a research‑grade peptide and is not intended for human or therapeutic use. At the concentrations used in kinase assays (10‑100 uM), the peptide is non‑toxic to cells. For animal studies, the compound is not directly administered; only the tissue lysates are assayed. Standard laboratory safety practices (gloves, lab coat, eye protection) should be used when handling the lyophilised powder or stock solutions.
Additional Infomation
Autocamtide‑2 amide has the peptide sequence KKALRRQEAVDAL (with the phosphorylation site typically at the serine within the RXXS/T motif; the exact sequence may vary by manufacturer). It is an amidated version of autocamtide‑2, with the C‑terminus amidated to prevent proteolysis. Autocamtide‑2 is widely used in neuroscience and cardiovascular research to study CaMKII‑dependent signalling pathways, including synaptic plasticity, neuronal excitability, cardiac contractility and hypertrophy. It is often used in conjunction with the inhibitory peptide AIP to confirm CaMKII‑specific activity.
These protocols are for reference only. InvivoChem does not independently validate these methods.
Physicochemical Properties
Molecular Formula
C65H119N23O19
Molecular Weight
1526.78
Appearance
White to off-white solid powder
HS Tariff Code
2934.99.9001
Storage

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.
Shipping Condition
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
Solubility Data
Solubility (In Vitro)
H2O :~50 mg/mL (~32.75 mM)
Solubility (In Vivo)
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

Injection Formulations
(e.g. IP/IV/IM/SC)
Injection Formulation 1: DMSO : Tween 80: Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution 50 μL Tween 80 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO 400 μLPEG300 50 μL Tween 80 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).
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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C,1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO 100 μLPEG300 200 μL castor oil 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10: 10 : 80 (i.e. 100 μL Ethanol 100 μL Cremophor 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH 400 μLPEG300 50 μL Tween 80 450 μL Saline)


Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).
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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders


Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

 (Please use freshly prepared in vivo formulations for optimal results.)
Preparing Stock Solutions 1 mg 5 mg 10 mg
1 mM 0.6550 mL 3.2749 mL 6.5497 mL
5 mM 0.1310 mL 0.6550 mL 1.3099 mL
10 mM 0.0655 mL 0.3275 mL 0.6550 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

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An example of molarity calculation using the molarity calculator is shown below:
What is the mass of compound required to make a 10 mM stock solution in 5 ml of DMSO given that the molecular weight of the compound is 350.26 g/mol?
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  • The answer of 17.513 mg appears in the Mass box. In a similar way, you may calculate the volume and concentration.

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What volume of a given 10 mM stock solution is required to make 25 ml of a 25 μM solution?
Using the equation C1V1 = C2V2, where C1=10 mM, C2=25 μM, V2=25 ml and V1 is the unknown:
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  • The answer of 62.5 μL (0.1 ml) appears in the Volume (Start) box
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Note: Chemical formula is case sensitive: C12H18N3O4  c12h18n3o4
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In vivo Formulation Calculator (Clear solution)
Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)
Step 2: Enter in vivo formulation (This is only a calculator, not the exact formulation for a specific product. Please contact us first if there is no in vivo formulation in the solubility section.)
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Calculation results

Working concentration mg/mL;

Method for preparing DMSO stock solution mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.

Method for preparing in vivo formulation:Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.

(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
             (2) Be sure to add the solvent(s) in order.

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